Rat neuro/glioblastomas induced by transplacental injection of ethylnitrosourea carry an oncogene that is detectable by transfection into mouse NIH 3T3 cells. (Shih et al., Nature (London) 290: 261-264 (1981), Schubert et al., Nature (London) 249: 224-227 (1974)). This gene was designated neu. (Schechter et al., Nature (London) 312: 513-516 (1984)). It was found that the neu gene was related to, but distinct from, the gene that encoded the epidermal growth factor receptor (EGFR). The transfected NIH 3T3 cells displayed a novel 185,000 dalton tumor antigen (p185) that was not. detected when the recipient cells were transformed by other oncogenes. (Padhy et al., Cell 28: 865-871 (1982)).
The human homolog of the rat neu oncogene has been isolated and termed c-erbB-2 or HER-2 on the basis of its close relationship to the human EGF receptor gene (also known as the c-erbB-1 gene). (Yamamoto et al., Nature 319: 230-234 (1986), Coussens et al., Science 230: 1132-1139 (1985)). The human neu protein has been reported to have a slightly higher apparent molecular weight of about 190,000 daltons. (Gullick et al., Int. J. Cancer 40: 246-254 (1987)). The DNA sequences of the rat and human clones which have been isolated predict a 1260 amino acid protein product of the neu gene that is colinear and about 50% identical with the predicted amino acid sequence of the EGF receptor. The level of sequence homology of the extracellular domains of the human EGFR and the c-erbB-2 is about 43%.
HER-2/neu differs from EGFR in that it is found on band q21 of chromosome 17 whereas the EGFR gene is located of band p11-p13 of chromosome 7. The HER-2/neu gene generates a messenger RNA (mRNA) of 4.8 kb which differs from the 5.8-10 kb transcripts for the EGFR gene. Finally, the protein encoded by the HER-2/neu gene has been found to have a molecular weight in the range from about 185,000 daltons to about 190,000 as compared to the 170,000 dalton protein encoded by the EGFR gene.
By analogy to the EGFR, the neu gene product appears to be a transmembrane protein consisting of a cysteine-rich extracellular region of about 650 amino acids, a transmembrane domain, and an intracellular portion of about 580 amino acids consisting in part of a tyrosine kinase domain.
Biochemical studies have revealed that the p185 protein is glycosylated and accessible to antisera in intact cells, consistent with its localization at the cell surface. p185 appears to be the receptor for an as yet unidentified ligand.
While a single point mutation occurring in the transmembrane domain of the protein converting a valine residue to glutamic acid was responsible for activation of the rat neu oncogene, no such mutation has been found to occur in the human neu gene. It is statistically unlikely that such a mutation would occur in the human homolog because two adjacent nucleotide changes would be needed to generate the same mutation in the transmembrane domain of the human neu gene. However, when the double mutation is induced in the human homolog, oncogenic activity is induced. This does not preclude the possibility that other point mutations could activate the human homolog.
The oncogenic potential of the human neu gene can be achieved by a mechanism other than a point mutation. Unlike the rat neu gene which is non-transforming at any level of expression unless mutated, the human neu gene is transforming, in the absence of any mutation, when overexpressed. Alteration of control of expression can be accomplished by increased expression of the pre-existing gene or by increasing the number of copies of the gene (gene amplification). Gene amplification of c-erbB-2 has been identified in primary mammary adenocarcinomas as well as in a salivary gland adenocarcinoma. Researchers have found that the human neu gene is amplified relatively frequently in human breast cancer cell lines. neu was amplified 2 to greater than 20 times in 30% of breast tumors. The presence of neu amplification was a significant predictor of both overall survival time and time relapse. (Slamon et al., Science 235: 177-182 (Jan. 9, 1987)). Thus, these findings suggest the possibility that neu overexpression, whether due to amplification or to some other mechanism, contributes to neoplastic growth.
Because the human neu protein appears to be involved in human malignancy, researchers have tried to study its expression and structure in human tissues.
Berger et al., Cancer Research 48: 1238-1243 (1988), tried to correlate c-erbB-2 gene amplification and protein expression with lymph node status and nuclear grading as well as with axillary lymph node involvement. c-erbB-2 specific antibodies generated from a synthetic peptide corresponding to residues 1215-1255 of the c-erbB-2 open reading frame were used to analyze fifty one primary human breast tumors for amplification of the c-erbB-2 proto-oncogene.
Drebin et al., Nature 312: 545-548 (1984) describe the generation of monoclonal antibodies that react specifically with cell-surface determinants found on NIH 3T3 cells transformed by transfection with a group of rat neuroblastoma oncogenes (the rat neu oncogene).
Drebin et al., Cell 41:695-706 (July 1985), describe the rapid and reversible loss of both cell-surface and total cellular p185 of NIH 3T3 cells transformed with the rat neu oncogene which were exposed to monoclonal antibodies reactive with the rat neu gene product.
Drebin et al., Oncogene 2: 273-277 (1988) describe monoclonal antibodies reactive with distinct domains of the rat neu oncogene-encoded p185 molecule which exert synergistic anti-tumor effects in vivo.
Drebin et al., Oncogene 2: 387-394 (1988), describe monoclonal antibodies which bind cell surface domains of the rat neu gene encoded product.
Expression of the c-erbB-2 protein in normal and transformed cells was investigated by Gullick et al., Int. J. Cancer 40: 246-254 (1987) using antisera generated against two synthetic peptides from the predicted sequence of the human c-erbB-2 protein and a monoclonal antibody specific for the rat neu protein.
Similarly, Venter et al., The Lancet, ii, pages 69-72 (Jul. 11, 1987) describe amplification of the human proto-oncogene c-erbB-2 in 12 of 36 human breast tumors which was associated with increased levels of expression of the c-erbB-2 protein, measured by immunohistological staining and by Western blotting. Affinity-purified rabbit antibodies, raised to a peptide consisting of residues 1215-1225 of the open reading frame of the c-erbB-2 protein, were used in the immunohistological staining.
Tandon et al., Journal of Clinical Oncology, pages 1120-1128, Vol. 7, No. 8 (August 1989), describe a method using Western blot analysis to quantirate the HER-2/neu protein levels in 728 human breast tumor specimens for the potential prognostic significance. Rabbit polyclonal antiserum was generated against the carboxy terminal synthetic peptide of the HER-2/neu protein (residues GTPTAENPEYLGLDVPV from the deduced amino acid sequence) for use in the study.
Akiyama et al., Science, pages 1644-1646, Vol. 232 (Jun. 27, 1986), also described raising antibodies against a synthetic peptide corresponding to 14 amino acid residues at the carboxy terminus of the deduced amino acid sequence from the human c-erbB-2 nucleotide sequence. The antibodies were reported to immunoprecipitate a 185,000 dalton glycoprotein from MKN-7 adenocarcinoma cells.
Most recently, regulation of the tyrosine kinase activity of the epidermal growth factor receptor by a truncated receptor of 100 kilodaltons containing the EGF-binding site but not the kinase domain was reported by Basu et al. in Molecular and Cellular Biology, pages 671-677 (February 1989). It was described that structurally related receptor kinases, such as the platelet-derived growth factor receptor, the insulin receptor, and the neu receptor, were not inhibited by the truncated 100 kDa receptor.